Recombinant Human B3GalT5 His-tag Protein, CF

R&D Systems | Catalog # 10555-GT

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Key Product Details

  • R&D Systems CHO-derived Recombinant Human B3GalT5 His-tag Protein (10555-GT)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

CHO

Accession Number

Q9Y2C3.1

Applications

Enzyme Activity
View all B3GalT5 Proteins and Enzymes »

Product Specifications

Source

Chinese Hamster Ovary cell line, CHO-derived human B3GalT5 protein
Asn29-Val310, with a C-terminal 6-His tag

Purity

>90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

Asn29

Predicted Molecular Mass

34.7 kDa

SDS-PAGE

40-45 kDa, under reducing conditions.

Activity

Measured by its ability to transfer galactose from UDP-galactose to N-Acetyl-alpha -D-glucosamine.
The specific activity is >200 pmol/min/μg, as measured under the described conditions.

Scientific Data Images for Recombinant Human B3GalT5 His-tag Protein, CF

Recombinant Human B3GalT5 His-tag Protein SDS-PAGE.

2 μg/lane of Recombinant Human B3GalT5 His-tag Protein (Catalog # 10555-GT) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 40-45 kDa.

Formulation, Preparation, and Storage

10555-GT
Formulation Supplied as a 0.2 μm filtered solution in Tris and NaCl.
Shipping The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.

Background: B3GalT5

B3GalT5 is a glycosyltransferase in the beta-1,3-galactosyltransferase (B3GalT) family that contains 5 members. It is synthesized as type II membrane-bound glycoproteins in the Golgi apparatus (1). The enzyme catalyzes the synthesis of type 1 lactosamine structure (Gal beta 1-3GlcNAc) in type 1 Lewis antigens (Lea and Leb). Lea and Leb are elevated in gastrointestinal and pancreatic cancers (2). Sialylated Lea (Neu5Ac alpha 2-3Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta ), also known as CA19-9, is a tumor marker in the gastrointestinal tract (Stomach, colon, and pancrease) (3). Down-regulation of B3GalT5 will reduce the level of CA19-9 (4, 5). In contrast, beta-1,4-galactosyltransferases synthesize type 2 lactosamine structure (Gal beta 1-4GlcNAc) that is found in type 2 Lewis antigens (Lex and Ley). B3GalT5 is also known as SSEA-3 synthase and synthesizes the structure Gal beta 1-3GalNAc in stage-specific embryonic antigen-3 (SSEA-3) antigen (6). SSEA-3 plays a key role in identifying many types of mammalian cells with pluripotent and stem cell-like characteristics (7). It also catalyzes the transfer of Gal to GlcNAc-based acceptors with a preference for the Core-3 O-linked glycan GlcNAc beta 1-3GalNAc structure (8). The activity of this enzyme has been measured with a phosphatase-coupled method (9).

References

  1. Isshiki S. et al. (1999) J. Biol. Chem. 274:1249.
  2. Holgersson J and Löfling J. (2006) Glycobiology 16:584.
  3. Magnani, JL (2004). Arch. Biochem. Biophys. 426:122.
  4. Mare L. and Trinchera M. (2004) Eur. J. Biochem. 271:186.
  5. Zulueta, A.et al. (2014) FASEB J. 28:946.
  6. Zhou D. et al. (2000) J. Biol. Chem. 275:22631.
  7. Kuroda, Y. et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107: 8639.
  8. Zhou D. et al. (1999) Eur. J. Biochem. 263:571..
  9. Wu, Z.L. et al. (2011) Glycobiology 21:727.

Long Name

UDP-Gal:betaGlcNAc beta 1,3-galactosyltransferase, polypeptide 5

Alternate Names

B3Gal-T5, B3GalT-V, B3GalTx, B3T5, beta-1,3-Galactosyltransferase 5, beta3Gal-T5, GlcNAc-Beta-1,3-Galactosyltransferase 5, GLCT5, UDP-Gal:Beta-GlcNAc Beta-1,3-Galactosyltransferase

Entrez Gene IDs

10317 (Human); 93961 (Mouse); 288161 (Rat)

Gene Symbol

B3GALT5

UniProt

Q9Y2C3.1

Additional B3GalT5 Products

Product Documents for Recombinant Human B3GalT5 His-tag Protein, CF

Certificate of Analysis

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Product Specific Notices for Recombinant Human B3GalT5 His-tag Protein, CF

For research use only

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Protocols

View specific protocols for Recombinant Human B3GalT5 His-tag Protein, CF (10555-GT):

Materials
  •  Assay Buffer: 25 mM Tris, 150 mM NaCl, 10 mM MgCl2, 10 mM MnCl2, pH 7.5
  •  Glycosyltransferase Activity Kit  (Catalog # EA001)
  •  Recombinant Human B3GalT5 (rhB3GalT5) (Catalog # 10555-GT)
  •  Donor Substrate: UDP-Galactose (Sigma, Catalog # U4500), 10 mM stock in deionized water
  •  Acceptor Substrate: GlcNAc (N-Acetyl-alpha -D-glucosamine) (Millipore, Catalog # 1079), 1 M stock in deionized water
  •  96-well Clear Plate  (Catalog # DY990)
  •  Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute 1 mM Phosphate Standard provided by the Glycosyltransferase Activity Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of Assay Buffer for a 100 µM stock. This is the first point of the standard curve.
  2. Continue standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock in Assay Buffer. The standard curve has a range of 0.078 to 5 nmoles per well.
  3. Prepare reaction mixture containing 0.4 mM UDP-Galactose, 200 mM GlcNAc and 4 µg/mL Coupling Phosphatase 1 (supplied in kit) in Assay Buffer.
  4. Dilute rhB3GalT5 to 8 µg/mL in Assay Buffer.
  5. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
  6. Load 25 µL of 8 µg/mL rhB3GalT5 into empty wells of the same plate as the curve. Include a Control containing 25 μL of Assay Buffer.
  7. Add 25 µL of reaction mixture to all wells, excluding the standard curve.
  8. Seal plate and incubate at 37 °C for 20 minutes.
  9. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
  10. Add 100 µL of deionized water to all wells. Mix briefly.
  11. Add 30 µL of the Malachite Green Reagent B to all wells.  Mix and incubate sealed plate for 20 minutes at room temperature.
  12. Read plate at 620 nm (absorbance) in endpoint mode.
  13. Calculate specific activity:

      Specific Activity (pmol/min/µg) =

Phosphate released* (nmol) x (1000 pmol/nmol)
Incubation time (min) x amount of enzyme (µg)

     *Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control.

Per Reaction:

  • rhB3GalT5: 0.2 µg
  • Coupling Phosphatase 1: 0.1 µg
  • UDP-Galactose: 0.2 mM
  • GlcNAc: 100 mM

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